Wound Tissue Disorders
The changing patterns of the connective tissue matrix during growth, development, and repair following injury require a delicate balance between synthesis and degradation of collagen and proteoglycans. Under normal circumstances this balance is maintained, while in many diseased states it is altered, leading to an excessive deposition of collagen or to a loss of functional tissue.
Collagen is the major structural constituent of mammalian organisms and makes un a large portion of the total protein content of skin and other parts of the animal body. In humans, it is particularly important in the wound healing process and in the process of natural aging. Various skin traumas such as burns, surgery, infection and accident are often characterized by the erratic accumulation of fibrous tissue rich in collagen and having increased proteoglycan content. In addition to the replacement of the normal tissue which has been damaged or destroyed, excessive and disfiguring deposits of new tissue sometimes form during the healing process.
Although balanced scar formation and remodeling are essential processes in skin wound healing, disorders of excess scar formation remain a common and therapeutically refractory clinical problem. A hypertrophic scar is an excessive wound scar which by definition has grown in size beyond that required for normal wound healing. Hypertrophic scars can emerge from many wound types, such as from a burn or a sharp incision.
Hypertrophic scars generally result from an over-production of cells, collagen and proteoglycan [Linares, H. A. and Larson, D. L., Plast. Reconst. Surg., 62:589 (1978); Linares, H. A., Plast. Reconstr. Surg., 818-820 (1983)]. These scars more frequently occur among children and adolescents, suggesting that growth factors may influence the development of this type of scar. Hypertrophic scars are especially common in patients who have burns or wounds that heal by secondary intention. These scars, by definition, exceed normal wound healing, causing problems that range from aesthetic deformity to severe limitation of motion. Histologically, these lesions are characterized by randomly distributed tissue bundles consisting of uniaxially oriented extracellular matrix and cells.
In these scars, the over-production and compaction of collagen and proteoglycans [Shetlar, M. R. et al., Burns 4:14 (1977)] exceeds the proliferation of cells. These histological observations suggest that the lesions result from loss of the normal control mechanisms which regulate the synthesis of extracellular matrix during would healing [Shetlar. M. R. et al., Burns 4:14 (1977)]. They are more common on the anterior surfaces of the neck, the shoulder, the chest wall and, in general, the flexor surfaces of the extremities. While some hypertrophic scars will spontaneously resolve within a few years, in many instances, especially in the locations mentioned above, they persist indefinitely. Because these scars are so common, particularly in burns or wounds that heal by secondary intention, their management represents a major unsolved clinical problem.
Keloids are tumors or connective tissue consisting of highly hyperplastic masses which occur in the dermis and adjacent subcutaneous tissue in certain susceptible individuals, most commonly following trauma. Keloid scars are a more severe form of hypertrophic wound scar and form firm dermal nodules of scar which are most commonly preceded by trauma at the site of origin. They are generally larger, grow in an apparently unregulated way, and tend to invade normal tissue surrounding the wound. They are commonly found on the face, earlobes and the medial surface of the ear. The size and growth of these scars often bear little relationship to the magnitude of the skin injury which led to their formation. Keloids often begin as a scratch or an acne pustule, but can grow and become very disfiguring. They rarely resolve spontaneously and often recur even after surgical intervention. Although keloid scars occur in all races, they are more common in African American and Asian populations and in females than in males. The known therapies for keloids have had limited success and they frequently can recur in the site after surgical removal.
The histologic features of disorders of excessive wound healing have been well described. They are characterized by randomly distributed tissue bundles of uniaxially oriented extracellular matrix and cells. Compared to normal scars, the cell density of these scars is low, reflecting the over-production of collagen and proteoglycans relative to the rate of cell proliferation, which is in conformance with the hypothesis that hypertrophic disorders result from the loss of normal control mechanisms that regulate the synthesis of extracellular matrix during wound healing. Fibroblasts harvested from hypertrophic scars, however, have not been found to be phenotypically abnormal. Hypertrophic scars appear to manifest the influence of epigenetic factors such as mechanical tension on the growth and biosynthetic processes of connective tissue cells. Keloids, on the other hand, result from an abnormal fibroblast phenotype and may therefore be classified as a genetic abnormality.
Scar Formation
The ability to heal by forming scars is essential for mammalian systems to survive wounding after injury. Normally, wound healing is a continuous process extending over a one-to-two-year period. Conceptually, the wound healing process may be divided into three phases, as illustrated in FIG. 1. The first (stage I) is an intensely degradative phase called the inflammatory stage. It occurs immediately after injury and provides a means to remove the damaged tissues and foreign matter from the wound. This phase lasts approximately one week. Two-to-three days later, as fibroblasts from the surrounding tissue move into the wound, the repairing process enters its second stage (stage II), the proliferation and matrix synthesis stage. The fibroblasts in the wound proliferate and actively produce macromolecules, such as collagen and proteoglycans, which are secreted into the extracellular matrix. The newly-synthesized collagen fibrils are cross-linked by lysyl oxidase and provide structural integrity to the wound. During this stage, fibroblasts also contract the intact collagen in order to reduce the surface area of the wound. This second stage lasts about three weeks. Hypertrophic scars usually appear at this stage.
In the final, remodeling phase (stage III), the previously constructed and randomly organized matrix is remodeled into an organized structure which is highly cross-linked and aligned to maximize mechanical strength. Natural skin wrinkles (relaxed skin tension lines) which align themselves in the direction of mechanical tension and become permanent on the face over time are a common manifestation of this control process. With hypertrophic scars and keloids, the biosynthetic phase continues longer than necessary to repair the wound. In order to maintain nutrient supply in these scars, vascular in-growth occurs, resulting in a large, highly vascularized scar which is unsightly and can be disabling.
Enzyme Action in Collagen Degradation
Enzymes are proteinaceous substances which act as catalysts for biological reactions, in some cases hydrolysis reactions and in others oxidation-reduction processes. Some enzymes have broad activity and others, such as collagenase (Clostridiopeptidase A) produced from the bacterium Clostridium hystolyticum, have very specific activity. Highly purified collagenase has been prepared and been found uniquely capable of cleaving bonds in the collagen structure permitting other enzymes to act on the resulting molecular fragments.
The first animal enzyme capable of degrading collagen at neutral pH was isolated from the culture fluid of tadpole tissue. This was shown to cleave the native molecule into two fragments in a highly specific fashion at a temperature below that of denaturation of the substrate. These fragments were characterized by electron microscopy and shown to reflect the cleavage of a native collagen molecule at a specific site closer to the C-terminal end of the molecule, yielding segments of one quarter and three quarters the length of the native collagen molecule; the larger fragment was termed TCA and the smaller fragment TCB.
Collagenolytic enzymes have been obtained following cell and organ culture from a wide range of tissues from animal species in which collagen is present. In general, these enzymes have a number of fundamental properties in common; they all have neutral pH optima; they are not stored within the cell, but, rather, appear to be secreted either in an inactive form or bound to inhibitors. FIG. 2 summarizes schematically the fundamental aspects of this enzyme and its mode of action. They appear to be zinc metalloenzymes requiring calcium, and are not inhibited by agents that block serine or sulphydryl-type proteinases. They are inhibited by kelating agents such as EDTA., 1.10-o-phenanthroline, and cysteine, which may inactivate zinc and perhaps other metals required for enzymatic activity and the zinc in the latent enzyme can be replaced by other divalent cations such as Co, Mn, Mg, and Cu. Nearly all the collagenases studied so far have a molecular mass that ranges from 25,000 to 60,000 daltons. The enzymes are usually present in a latent or inactive form. In some instances they seem to be associated with the presence of a zymogen, but in most cases are bound to an inhibitory protein component that can be removed to form the active enzyme; this step is accompanied by a decrease in molecular weight. Although proteolytic enzymes have been mostly used for activation, some latent collagenases can be activated by nonproteolytic agents, such as cheotropic salts or organic mercurial compounds, suggesting that the collagenase and inhibitors, though forming a tight complex, might not be peptide linked as in a proenzyme.
The cells that synthesize collagenase are influenced to a great extent by the environment in which they live. This includes the permanent resident cells of the connective tissues and adjacent structures and the migratory cells that accumulate as a result of injury, inflammation, or immune phenomena, as well as the products secreted by these cells. Epithelial cells and factors secreted by such cells may also play a significant role in the development and remodeling of connective tissues by virtue of their ability to regulate collagenase production by the mesenchymal cells.
Mammalian collagenases display a great deal of specificity by hydrolyzing a single polypeptide bond on each chain of the native triple-stranded collagen helix. A significant amount of work has been devoted to understanding the unique characteristics of the cleavage site. The cleavage site of the .alpha.-1 chain was identified by electron microscopy and later by sequence analysis.
Although no conclusive physicochemical explanation is available to describe the specificity of the cleavage, there are indications that the helix in that region is thermodynamically less stable. The actual bond cleaved in all species studies is a Gly-Leu or Gly-Ile link. There are slight differences in the amino acid sequence surrounding the scission site; these may account for the differences in the rates at which various collagens are degraded.
The binding of human skin fibroblast collagenase to reconstituted collagen has been recently studied in detail. The enzyme interacts tightly with the collagen fiber and appears to remain bound to the macromolecular aggregate during the degradation process. Approximately 10% of the collagen molecules in the reconstituted collagen appears accessible for binding, in close agreement with the theoretical number of molecules estimated to be present on the surface of the fiber. The in vitro data obtained seemed to indicate that digestion proceeds until completion without the enzyme returning to the solution, but, rather, hopping from one molecule to another. These observations also would explain why collagenase activity is enhanced when the enzyme is exposed to dilute suspensions of re-constituted polymeric collagen rather than compact fibers of large diameter. This mode of action of the enzyme, and its modality of handling the substrate, may explain the low turnover numbers observed (25 molecules of collagen cleaved per enzyme per hour), one of the lowest turnover numbers associated with an enzymatic reaction. It is possible that in vivo the rates could be even slower, due to the presence of enzyme inhibitors and because the active enzyme may have to compete with the latent enzyme for binding sites on the collagen fiber.
Another factor that seems to slow the breakdown of collagen is the presence of cross-links. The introduction of artificial methylene bridges with formaldehyde or of native cross-links by the use of purified lysyl oxidase increases the resistance of collagen to collagenase degradation. Native collagen fibers cross-linked by glutaraldehyde cannot be digested even by bacterial collagenase. Collagen from individuals of increasing age becomes more resistant to enzymatic digestion, suggesting that an age-related accumulation of cross-links may be responsible. It is, therefore, possible that cross-linking of collagen plays a role, not only in generating mechanical stability to the fibers, but also in the regulation of collagen turnover in vivo.
Scar Treatment
Existing therapy for hypertrophic scars and keloids includes surgery, mechanical pressure, steroids, x-ray irradiation and cryotherapy. There are many disadvantages associated with each of these methods. Surgical removal of the scar tissue is often incomplete and can result in the development of hypertrophic scars and keloids at the incision and suture points. Steroid treatments are unpredictable and often result in depigmentation of the skin. X-ray therapy is the only predictably effective treatment to date; however, because of its potential for causing cancer, it is not generally recommended or accepted. The most common approach to control hypertrophic scar and keloid formation is to apply pressure, which appears to be effective in many instances. However, this treatment has limited application, generally based on the size and location of the scar tissue on the body. Other commonly used treatments are application of Vitamin E and corticosteriods. Each of these agents can interfere with collagen synthesis and promote collagen degradation.
An additional method that has been observed empirically to result in general improvement in the appearance and size of treated scars involves covering the scar surface with a wound dressing fabricated from a silicone-based gel [Quinn, K. J., et al., Burns 12, 102-108 (1985); Quinn, K. J., Burns 13, S33-S40 (1987); Mustoe, T. A., et al., Surgery 106, 781-787 (1989).] Quinn et al. used a gel, such as that available from Dow Corning, Arlington, Tex., marketed under the name SILASTIC.RTM. gel, and as further described in U.S. Pat. Nos. 4,991,574 assigned to Dow, and 4,838,253 assigned to Johnson & Johnson, herein incorporated by reference.
Silicones are a group of completely synthetic polymers containing the recurring group --SiR.sub.2 O--, wherein R is a radical such as an alkyl, aryl, phenyl or vinyl group. The simpler silicones are oils of very low melting point, while at the other end of the scale of physical properties are highly cross-linked silicones which form rigid solids. Intermediate in physical properties are silicone elastomers such as gels and rubbers. A variety of such silicone gels have seen used as wound dressings, as disclosed in U.S. Pat. Nos. 4,838,253 to Brassington, and No. 4,991,574 to Pocknell.
Based on experiments involving the measurement of physical parameters associated with the use of such gels, investigators have concluded that the mode of operation of the silicone gel in scar treatment did not involve pressure, temperature, oxygen tension, or occlusion. Rather, as reported, the likely mechanism involved both hydration of the stratum corneum and the release of a low molecular-weight silicone fluid from the gel.
Subsequent investigations by Quinn (1987) and Mustoe, et al. (1989) confirmed the earlier conclusions that neither pressure, temperature, nor oxygen tension could account for the mode of action of the silicone gel in scar treatment, conclusions that remain widely accepted today. Mustoe, et al. (1989) differed from Quinn (1985, 1987) in concluding that a silicone-based chemical interaction was not likely, and in proposing a mechanism based on occlusion of the scar tissue by the silicone gel.
The use of collagenase in medical practice is well known but has heretofore been limited to topical application for debridement of dermal ulcers and burns and, recently, for the treatment of prolapsed intervertebral discs. Purified collagenase has been demonstrated to be relatively safe even in large doses (thousands of units) in animals and in contact with human blood vessels, nerves and bones. U.S. Pat. No. 4,645,668 to Pinnell et al. discloses a method for the treatment of scars involving the injection of a variety of enzymes, some combined with the enzyme collagenase, directly into existing scar tissue. Also disclosed is a method for the prevention of scar formation involving the administration of various enzymes, either alone or in combination, to surgically or traumatically-induced wounds.
U.S. Pat. No. 5,132,119, incorporated herein by reference, has disclosed that calcium antagonists in various forms can drive the cells toward extracellular degradation instead of biosynthesis in the tissue culture environment. Calcium antagonists appear to influence cells to assume a more spherical shape, a result illustrated in FIG. 3. These fibroblasts will concomitantly change their metabolic status from one of synthesis to one of degradation. They also produce considerably more collagenase than the same cell in a more spread configuration, indicating that agents which depolymerize cytocellular proteins inhibit collagen synthesis and accelerate the activity of collagenase. Thus, the factors which control fibroblast shape also control the dynamic balance between extracellular matrix and degradation.
Despite the various treatments presently available, there has been no widely accepted and predictably effective means for preventing or treating hypertrophic scars or keloids.